Genetics, Development and Cell Biology Ph.D. candidate: Kevin Natukunda

Major: Genetics

Major Professor: Julie Kuhlman

Title: "From the fish tank to the human gut: Characterization of the role of kita and ano1 ICC markers in regulation of intestinal motility in the zebrafish"

Abstract: Regulation of gut motility is a very complex process and is thought to require proper performance, regulation, functioning and coordination of three major cell types: the smooth muscle cells, the enteric nervous system and a specialized group of cells called the interstitial cells of Cajal (ICC).  The ICC, also commonly called the pacemaker cells of the gut have been implicated in proper gut motility. Reduction in the number, abnormal integrity or loss of ICC has been showed in patients with GI motility disorders such as Hirschsprung’s disease, slow transit constipation, gastroparesis, achalasia, and intestinal pseudo-obstruction. Although the role of ICC in GI motility has been widely examined at the phenotypic level, the underlying molecular mechanisms remain elusive. The studies presented in this dissertation utilized the revolutionary CRISPR-Cas9 mediated genome editing to generate reporter lines for two ICC biomarkers (kita and ano1) and characterize the expression pattern of kita and ano1 reporter lines using both confocal laser scanning microscopy and fluorescent microscopy. Gut motility in the (kita and ano1) null mutants was also characterized using both qualitative and quantitative approaches.     

The first study reports generation of kita and ano1 reporter lines (kita^is32gal4 and ano1^is33gal4) using the CRISPR-Cas9 mediated knock-in strategy into the zebrafish genome by homology directed repair mechanism. The generated reporter lines have a bright RFP expression and homozygous mutants are viable and can reproduce. This is the first study to successfully generate ano1 reporter lines in zebrafish.

In the second study, qualitative and quantitative approaches were used to examine the gut motility phenotypes of kita and ano1 null mutants at 9 dpf. The results indicated that zebrafish intestinal motility pattern was disrupted by knock-out of kita or ano1. There was a significant reduction in the average number of contractions, average distance traveled by individual contractions, frequency of the contractions, average time taken by individual contractions and the average interval between contractions. Our results suggest that both kita and ano1 are critical for generation and maintenance of coordinated, steady and complete contraction in the zebrafish intestine. We provide the first evidence for the role of ano1 in zebrafish gut motility using in vivo approaches and this novel finding will improve our present understanding of GI motility disorders.